The long-term goal of this project is to understand mechanisms of cardiac cell differentiation. The specific goals of this proposal are to identify genes important for cardiac valve formation in zebrafish and analyze their function. Cardiac valves are formed at several places along the vertebrate heart tube including the atrioventricular (AV) boundary which has been the focus of our studies. During the process of AV valve formation, myocardial cells at the AV boundary induce a subset of the underlying endocardial cells to undergo an epithelial to mesenchymal transition (EMT) and form an endocardial cushion. This cushion is later remodeled into a valve. Studies in chick and mouse embryos have led to the identification and analysis of several molecules important for distinct steps in valve formation but clearly additional molecules critical for this process remain to be identified. Previous genetic screens in zebrafish have led to the identification of two mutations that affect valve formation, jekyll and cardiofunk. We have recently isolated the Jekyll gene through a positional cloning approach and found that it encodes UDP-glucose dehydrogenase, an enzyme that primes sugars to be added into glycosaminoglycan (GAG) chains. GAG chains are the building blocks of Hyaluronic acid and are also covalently linked to proteoglycans. In order to understand the role of jekyll and cardiofunk in regulating valve formation, we propose the following specific aims: 1) further analyze their valve phenotype through histological analyses, gene and protein expression studies, and cell transplantation experiments; 2) utilize a transgenic line, generated in our lab, that expresses GFP in all endothelial, including endocardial, cells to examine valve formation in real time both in wild-type and in mutant embryos; and 3) map cardiofunk genetically and initiate its isolation by a positional cloning approach. We will also conduct large-scale genetic screens in diploid embryos to identify additional mutations affecting cardiac valve formation. We will begin to analyze the mutations defining new loci in order to dissect the role these genes play in cardiac valve formation. These molecular genetic studies of cardiac valve formation should lead to the identification of critical regulators of this process as well as to a detailed understanding of their function. This information should enhance our understanding and ability to diagnose, and possibly correct, valve and septation defects in humans.